RNA Interference Screening to Identify Novel Therapeutic Targets

RNA interference (RNAi) technology provides a powerful tool to study the function of genes and their encoded proteins and has opened up exciting avenues towards therapeutic target discovery.

To test the RNAi screening methodology in high-risk childhood sarcoma, we have developed a small-interfering RNA (siRNA) screening assay in two Ewing sarcoma cell lines. In Ewing sarcoma, as in many other cancers, the insulin-like growth factor-1 receptor (IGF1R) is emerging as therapeutic target and IGF1R blocking antibodies show promising anti-tumor activity in some but not all patients. So a key question is as to what underlies IGF1R inhibitor sensitivity versus resistance. We therefore applied the siRNA assay to screen the IGF1R signalling axis and all known protein tyrosine kinases (PTK) in the presence and absence of an IGF1R inhibitor.

This strategy revealed that Ewing sarcoma and rhabdomyosarcoma cells are exquisitely sensitive to loss of distal IGF1R signalling components such as the ribosomal protein S6 (RPS6); and IGF1R inhibitors failed to block RPS6 activation in resistant cell lines. The synthetic lethality approach revealed that siRNA knock-down of certain PTK such as the MET family receptor MST1R restores inhibitor efficacy, even in highly drug-resistant cell lines. Moreover, we confirmed MST1R expression in childhood sarcomas and found that MST1R loss blocks downstream RPS6 activation when combined with IGF1R inhibition in vitro.

These data identify MST1R as a potential therapeutic target, underscore the importance of understanding PTKs as signalling networks for successful implementation of kinase inhibitor strategies and provide a proof-of-principle for siRNA screening approaches in Ewing sarcoma.